This application for the Mentored Clinical Scientist Development Award (K08) seeks support for Ardath Yamaga, M.D., who recently completed her clinical fellowship in Pediatric Gastroenterology and Nutrition and is joining the faculty at the University of Southern California Keck School of Medicine. Hepatitis C Virus (HCV) is estimated to affect 4 million people in the United States, approximately 2% of the population, including children. Up to 80% of those infected with HCV become chronic carriers and have an increased risk of cirrhosis, hepatocellular carcinoma and liver-related deaths. Under the mentorship of Jing-Hsiung James Ou, Ph.D., Dr. Yamaga, will continue to pursue her investigations into the molecular biology and the pathogenesis of HCV. The proposed research is focused on the HCV NS2 protein. This protein, as well as a portion of its adjacent NS3 sequence, contains a metalloprotease activity that cleaves the NS2-NS3 junction. Preliminary studies of Dr. Yamaga indicate that NS2 has multiple signal sequences and multiple transmembrane domains. This poses an interesting question regarding how NS2 mediates the cleavage of the NS2-NS3 junction. The first aim of the proposed research is to continue to investigate the molecular mechanism that regulates the membrane translocation of NS2 for the purpose of understanding its membrane topology. This membrane topology will then be used as a guide in Specific Aim 2 to study the molecular mechanism that mediates the cleavage of the NS2-NS3 junction. The hypothesis that the catalytic domain resides in the N-terminus of NS3 and that this protease domain needs to interact with the cytosolic domains of NS2 for activation will be investigated. NS2 is not required for the replication of HCV RNA. On the contrary, it interacts with the HCV envelope proteins. This raises the possibility that NS2 may be involved in viral morphogenesis and may even be a component of the virion. Thus, the third aim of the proposed research is to investigate how NS2 interacts with HCV envelope proteins and whether and how NS2 interacts with cellular proteins. The goal of this part of the research plan is to understand the biological functions of the NS2 protein. Under Dr. Ou's mentorship and with support of this award, Dr. Yamaga will acquire new knowledge and skills in the sciences of molecular biology and viral pathogenesis and develop her independent research career.